Analytical and diagnostic procedures are crucial in many functions in the modern society. One of the most common is the diagnostic procedures performed at health care institutions (e.g. hospitals) with the purpose to determine if a patient has a particular disease. For example, elevated concentration of the prostate specific antigen (PSA) in male blood is an indication of ongoing prostate cancer in the patient. However, this is not specific enough and there is need for better methods to analyze different modifications of PSA to improve the clinical decision based on analytical results. Other analytical or diagnostic procedures include, but are not limited to, the diagnosis of cancer based on ocular analysis of stained tissue biopsies, diagnostics of cattle prior to slaughter in order to produce safe food, diagnostic procedures in veterinary sciences with the purpose of treating sick animals, targeted radiotherapy of tumors in animal or humans, the detection of pathogens or toxins in food or feed stuff, the determination of the concentration of nutritional supplements (e.g. vitamins) in processed food or feed stuff, the detection of hazardous chemicals in the environment and the like.
Whenever the solid object structure is complex, most of the currently used methods must use reagents that are highly specific to amplify the signal from one component in the complex sample. Such complex structures can be cell surfaces on cells in tissue or in body fluids. Still other types of complex structures are or protein complex where the individual proteins change conformation or can be modified by posttranslational modifications.
One particular method for diagnosis is immunohistochemistry (IHC). Diagnostic IHC procedures are developed for a multitude of diseases, most notably for cancers. In brief, IHC is a method wherein a thin slice of tissue is placed onto a microscope glass slide followed by staining of selected receptors. An image is made of the stained tissue slice and a trained operator is judging if the tissue contains staining patterns indicative of disease. Even though IHC is used world-wide and has improved the possibility to diagnose serious diseases like cancer, general IHC methodology still suffers from poor repeatability and long tissue preparation protocols (as evident in the report “Current issues in ER and HER2 testing by IHC in breast cancer”. by Allen M Gown published in Modern Pathology 2008 May; 21 Suppl 2:S8-S15, which is incorporated by reference herein).
Immunohistochemistry is one of the dominant methods for analysis of tissue slices. Being used in the majority of major hospitals, it is a well known method for persons skilled in the art of tissue analysis. In brief, IHC is a method for localizing proteins in cells of a tissue section by use of antibodies binding specifically to antigens in biological tissues. IHC staining is commonly used in the diagnosis of abnormal tissue such as tumors. Specific molecular structures on or in the cells in the tissue are characteristic of particular cellular events indicative of disease. In order to visualize the antibody-antigen interaction, the antibody can be tagged to a fluorophore, such as FITC, rhodamine, Texas Red or any other fluorescent moiety. In the procedure thin (typically 20 μm) slices are taken of the tissue of interest. The tissue is then treated to rupture the membranes, usually by using a detergent (e.g. Triton X-100). After these steps, the tissue slice is prepared for antibody treatment, which typically follows an indirect approach. The indirect approach involves a primary (unlabeled) antibody which reacts with tissue antigen, and a secondary (labeled) antibody which reacts with the primary antibody. The secondary antibody is normally labeled with a fluorescent moiety or an enzyme. IHC is a powerful detection technique and is capable of showing exactly where a given protein is located in the tissue sample. IHC is widely used in many fields of biology, e.g. in the neurosciences, enabling researchers to examine protein expression within specific brain structures and in diagnostic surgical pathology for typing tumors (e.g. carcinoma vs melanoma). The result of an IHC analysis is always an image of the tissue slice with areas containing certain targeted receptors stained in a distinguishable color. As such, IHC is an end-point measurement, i.e. it is only possible to detect the status of the antibody-antigen interaction at one point in time. IHC also suffers from the often manual interpretation of images; trained operators may disagree on the extent and intensity of the staining of the very same tissue slide, leading to uncertainties when comparing results across operators and laboratories. Another major disadvantage of IHC is that it is impossible to show in IHC that the staining corresponds with the protein of interest. A description of IHC used in clinical practice is available in the report “Diagnostic evaluation of HER-2 as a molecular target: an assessment of accuracy and reproducibility of laboratory testing in large, prospective, randomized clinical trials”. By Press M F, Sauter G, Bernstein L, Villalobos I E, Mirlacher M, Zhou J Y, Wardeh R, Li Y T, Guzman R, Ma Y, Sullivan-Halley J, Santiago A, Park J M, Riva A, Slamon D J. Published in Clinical Cancer Research 2005 Sep. 15; 11(18):6598-607, which is incorporated by reference herein.